Connexins (Cxs) are membrane proteins constituting the gap junctions (GJs), which provide a regulated conduit for intercellular ionic (e.g., K+, Ca++) and biochemical (e.g., nutrients and signaling molecules) couplings. Genetic studies have linked more than 100 mutations in Cx genes to a large proportion of prelingual nonsyndromic deafness. Despite their importance in hearing, we know very little about the role Cxs play in the cochlea. Our long-term goal is to understand the molecular mechanisms maintaining homeostasis in the mammalian cochlea, especially the role played by the Cx family of proteins in the cochlea. In this 5-year project, we plan to conduct three series of experiments utilizing a multidisciplinary approach to test the HYPOTHESIS that biochemical coupling mediated by heteromultimeric GJs assembled from Cx26 and Cx30 in the cochlea is required for normal hearing in mice. First, we will use an in vitro system to investigate whether some human Cx26 mutations linked to deafness specifically affect biochemical permeability of cochlear GJs. Since most cochlear GJs are constituted by heteromultimeric assembly of Cx26 and Cx30, therefore deleting one Cx gene does not necessarily eliminate GJ channels in the cochlea. Our second series of experiments will investigate whether GJ channels are still functional for ionic permeation in the cochlea of Cx30-/- mice. Using in situ preparations and biochemical assays, we will test whether a change in the molecular configuration of wild type GJs are responsible for deafness in Cx30-/- mice. In the last series of experiments we will rescue the hearing of Cx30-/- mice by transgenic expressions of either Cx30 or Cx26 under the spatial and temporal controls of the Cx30 gene regulatory mechanisms integrated in bacterial artificial chromosome. These genetically reconstituted GJs in Cx30-/- mice consisting of native or non-native mixings of wild type Cxs mainly affect intercellular biochemical couplings. Results obtained from the third series of experiments will further test our hypothesis in vivo. Design of any effective treatment for patients suffering from sensorineural hearing loss caused by Cx mutations depends on our understanding of the role Cxs play in the cochlea. With successful implementation of the specific aims proposed here, we hope to significantly advance our knowledge of intercellular communication mediated by GJs in the cochlea and improve our ability to help millions of deaf patients in the future.